[Cancer Biology & Therapy 6:12, 1938-1943; December 2007]; 2007 Landes Bioscience

Research Paper

Green Tea Polyphenol and Epigallocatechin Gallate Induce Apoptosis

and Inhibit Invasion in Human Breast Cancer Cells
Rajesh L. Thangapazham1,2
Neena Passi1
Radha K. Maheshwari1,*
1Department

of Pathology; Uniformed Services University of the Health Sciences;

Bethesda; Maryland USA
2Birla Institute of Technology and Science; Pilani, India

*Correspondence to: Radha K. Maheshwari; Department of Pathology; Uniformed

Services University of the Health Sciences; 4301 Jones Bridge Road; Bethesda,
Maryland 20814 USA; Tel.: 301.295.3394; Fax: 301.295.1640, Email: rmaheshwari@
usuhs.mil
Original manuscript submitted: 06/27/07
Manuscript accepted: 09/01/07
Previously published as a Cancer Biology & Therapy E-publication:
http://www.landesbioscience.com/journals/cbt/article/4974

Key words
green tea, EGCG, MMP-9, AKT, apoptosis,
invasion, MDA-MB-231
Acknowledgements
This work was supported by intramural grant
from the Uniformed Services University of
the Health Science, Bethesda and US-INDIA
Foreign Currency Fund from US Department
of State to USUHS. The authors are grateful
to Mr. Anuj Sharma for his help and valuable suggestions. The opinions or assertions
contained herein are the private views of the
authors and should not be construed as official or necessarily reflecting the views of the
Uniformed Services University of the Health
Sciences or the Department of Defense, USA.

1938

Abstract
Currently, there is no effective therapy for estrogen independent breast cancer. MDAMB-231 is an estrogen receptor negative highly invasive human breast cancer cell line
and has been used as a relevant model system to evaluate drugs with chemopreventive
potential against highly invasive breast cancer phenotypes. Epidemiological studies
though inconclusive have shown that consumption of Green Tea Polyphenols (GTP)
reduces the incidence and progression of breast cancer. Green tea is an important source
of antioxidants that may be useful for chemoprevention of cancer. Recently published
preclinical study from our lab suggested that GTP and EGCG treatment inhibit proliferation and induce apoptosis of MDA-MB-231. In this study, we have evaluated apoptotic
and anti-invasive activity of green tea polyphenols (GTP) and its principal constituent
Epigallocatechin gallate (EGCG) in MDA-MB-231 human breast cancer cell line. In in
vitro human breast cancer model, EGCG and GTP induced apoptosis and significantly
decreased invasion of breast cancer cells. Western blotting of MDA-MB-231 cell lysates
from EGCG and GTP treated and untreated control revealed an increase in bax, reduction in bcl2 and PARP cleavage. Quantitative fluorescence labeling resulted in a 2428%
reduction in invasion through matrigel by EGCG and 1523% reduction by GTP in
a dose dependent manner. Focussed microarray analysis and reverse transcriptase
polymerase chain reaction and zymogram analysis revealed inhibition of MMP-9 expression by polyphenol treatment. Furthermore, AKT was found to be inhibited both at the
RNA and protein level by polyphenol treatment. Moreover EGCG and GTP decreased
AKT phosphorylation as found out by Western blotting for Phospho-AKT (Ser-473).
beta-catenin level was found to be decreased both in cytoplasm and nucleus. For the first
time we report the connection of beta-catenin and AKT modulation by GTP and EGCG
as a possible mechanism for the induction of apoptosis in human breast cancer cells and
also inhibition in their invasive capacity.

Introduction
A report of breast cancer statistics for the years 1975 through 2003 estimates that
12.7%, meaning 1 in 8 of women born in United States today, will develop breast cancer
at some time in their lives.1 Premalignant progression and chronic administration of
frequently used hormone responsive breast cancer treatments like Aromatase inhibitors
and Specific Estrogen Receptor modulators may result in a hormone non-responsive
form of the disease.24 MDAMB231 is an estrogen receptor negative, highly invasive
human breast cancer cell line and has been used as a relevant model system to evaluate
chemopreventive agents against highly invasive and hormone non-responsive breast cancer
phenotypes.
Epidemiological studies suggest that increased consumption of green tea is also related
to improved prognosis of human breast cancer5 and the risk of breast cancer is found
to be less in AsianAmericans who consume green tea.6,7 This study investigated cancer
chemopreventive effects of green tea polyphenols (GTP) and the most abundant green
tea catechin, epigallocatechin gallate (EGCG). There are several reviews which discuss the
modulation of important signaling events, direct targets and gene expression by EGCG and
their implications in cancer chemoprevention.810 In recent studies, GTP and EGCG have
been shown to have therapeutic potential in treatment of cancers using MDAMB231 as
a model system. GTP and EGCG have been shown to suppress MDAMB231 tumors in
vivo11 and exhibit proapoptotic, antiinvasive and antiproliferative properties in vitro.11,12
In a previous study we have demonstrated the anti tumor effect of GTP and EGCG in
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Green Tea Induces Apoptosis and Inhibits Invasion

nude mice inoculated with human breast cancer MDAMB231 of Tris (pH 8), 10 mM of EDTA (pH 8) and were fractionated on
cells. Polyphenols were effective in delaying tumor incidence as well 1.5% agarose gel.
as in reducing the tumor burden when compared to water fed and
Western blotting. Cells were harvested, pelleted and were homogsimilarly handled controls. GTP and EGCG treatment was found enized with icecold homogenization buffer (50 mM TrisHCl, pH
to inhibit proliferation and induce apoptosis of MDAMB231 cells 7.4, 150 mM NaCl, 1% Triton X 100, 1% sodium deoxycholate,
invitro and invivo.
0.1% SDS to which 1 mM DTT, 1 mM PMSF, 1 mg/ml pepstatin A,
In the present study, we have investigated mechanisms involved 10 mg/ml Leupeptin, 10 mg/ml aprotinin, 25 mM NaF and 100 mM
in proapoptotic and antiinvasive effects of EGCG and GTP in Na3VO4 were added fresh). The homogenate was passed ten times
MDAMB231. The results showed that EGCG and GTP induced through a 25guage needle and centrifuged at 14,000 g for 20 min at
apoptosis and suppressed invasiveness of MDAMB231 cells in a 4C. The supernatant protein extract was transferred to fresh tubes,
dose dependent manner. A fragmented DNA ladder was detected by aliquoted and stored at 80C. Protein concentration was determined
electrophoresis in cells treated with polyphenols indicating apoptosis. using a BCA protein assay kit (Pierce, Rockford, IL). Fifty mg of
AKT, which is a serine/threonine kinase, regulates cell survival and protein extracts were Western blotted as described previously.11
invasion. Treatment of cells with varying concentrations of EGCG
Invasion assay. MDAMB231 was grown to 7080% confluence.
and GTP inhibited AKT at both RNA and protein level. Moreover Cell suspension were prepared by trypisinizing the monolayer and
EGCG and GTP treatment resulted in lesser phosphorylated AKT resuspending in medium without FBS at 5 x 104 cells/ml. BD Falcon
when compared to untreated controls. Polyphenol treatment in this HTS FluoroBlok 24Multiwell Insert plate system (catalog No.
cell line decreased the level of betacatenin in the cytoplasm and also 354165) was prepared by rehydrating BD Matrigel Matrix coating
reduced its accumulation in the nucleus. Our results suggest that the with PBS for 2 h at 37C. The rehydrating solution was carefully
downregulation of AKT plays an important role in proapoptotic and removed, 0.75 ml of medium containing 10% FBS (chemoattracantiinvasive potential of green tea. These studies have greater clinical tant) was added to the well and 0.5 ml of cell suspension was added
significance since the ability of polyphenol to activate apoptotic to each insert well. EGCG and GTP were added to the medium in
program and decrease invasiveness of tumor cells might determine both upper and lower chambers along with the cells and chemoatthe success of chemotherapy.
tractant solution. Uncoated insert plates BD Falcon HTS FluoroBlok
24Multiwell Insert system (catalog No. 351157) included as migration control, were used without rehydration. Following incubation
Materials and Methods
for about 24 h, the medium was removed from upper chamber and
EGCG and GTP were obtained from LKT laboratories (St. Paul, entire insert plate was transferred to a BD Falcon 24well plates
Minnesota). MDAMB231 breast cancer cell line was obtained for post cell invasion labeling (catalog no. 351147) containing 0.5
from ATCC (Rockville, MD). Antibodies for proteins involved ml/well of 4 mg/ml Calcein AM in Hanks buffered saline. The plates
in apoptosis were purchased from BD Biosciences, San Jose, CA. were incubated for 1 h at 37C and read in a fluorescence plate reader
Betacatenin antibodies and AKT antibodies were purchased from with bottom reading capabilities without further manipulation.
Santa Cruz Biotechnology, Santa Cruz, CA. PhosphoAKT (Ser473)
Isolation of RNA. Cells gown with or without GTP and
antibody was purchased from cell signaling technology, Danvers, EGCG trypsinised, pelleted and washed with PBS and transferred
MA.
to 1.5 ml microfuge tubes. Total RNA was extracted using TriZol
Cell lines. Estrogen receptornegative MDAMB231 was kit (Invitrogen life technologies, Carlsbad, CA) and quantitated
obtained from ATCC (Rockville, MD). Cells were maintained in spectrophotometrically using Beckman DU640 Spectrophotometer
monolayer in Eagles minimum essential medium supplemented (Beckman instruments Inc., Columbia, MD). RNA quality was
with 5% fetal bovine serum, nonessential amino acids, 2x vitamin determined by electrophoresis on 1% agarose formaldehyde gel.
solution, pencillin and streptomycin. Cell cultures were incubated at
Oligo GEArray microarray. Human Breast Cancer Biomarker
37C in a humidified atmosphere of 5% CO2 and 95% air and were Oligo GEArray Microarray kit (Superarray Bioscience Corporation,
maintained at 7090% confluence and medium was changed every Frederick, MD) was used and experiments were carried out as per
other day. Cells were dislodged for both passaging and harvesting the manufactures protocol. cRNA probe was synthesized from
by a brief incubation in 0.25% trypsin and .02% EDTA. Cells were 1 mg of RNA using TrueLabelingAMP Linear RNA Amplification
stained with trypan blue and counted using a hematocytometer.
Kit, SuperArray Bioscience Corporation, Frederic, MD. The ampliDNA fragmentation. To measure DNA fragmentation, 1 x 106 fied cRNA was purified using spin column (ArrayGrade cRNA
untreated or treated MDAMB231 cells were washed with PBS and Cleanup Kit, SuperArray Bioscience Corporation, Frederic, MD)
then resuspended in 10 mM of Tris (pH 8), 10 mM of EDTA (pH and quantitated spectrophotometericaly. Array membranes were
8). Proteinase K was added to a final volume of 200 mg/ml followed prehybidized for 2 hr/60C/1020 rpm hybridization solution
by 10% SDS to obtain a final concentration of 0.5%. Samples were followed by overnight hybridization with 4 mg of cRNA mixed in
incubated at 55C for 4 h. 5 M NaCl was added to a make a final 750 ml of hybridization buffer at 60C/1020 rpm in hybridizaconcentration 1 M and incubated on ice for 45 min. Protein precipi- tion chamber. Array membranes were then washed for 15 min each
tate were sedimented by centrifugation at 5000 g for 15 min and at 60C/1020 rpm with pre-warmed wash solution 1 (2XSSC,
the supernatant was transferred to a fresh tube. DNase free RNase 1% SDS) and wash solution 2 (0.1XSSC, 0.5%SDS) respectively.
was added to a final concentration of 25 mg/ml and was incubated Blocking was done for 2 hr/1020 rpm at room temperature (RT)
at 37C for 60 min. DNA was precipitated overnight a 20C after with 2 ml of blocking solution Q followed by incubation with
adding 2 volumes of 80% ethanol. DNA was pelleted by centrifuging 2 ml of binding solution (1:15000 Apstrep in 1xBuffer F). Array
at 10,000 g for 30 min at 4C. The pellet was resuspended in 10 mM membranes were then washed with 1X buffer F and rinsed with
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Cancer Biology & Therapy

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Green Tea Induces Apoptosis and Inhibits Invasion

Figure 1. Effect of EGCG and GTP on DNA fragmentation. Representative

picture from three independent experiment of DNA fractionation
from MDAMB231 treated with 50 and 80 mg/ml of EGCG and 40 and
60 mg/ml GTP for 24 hrs.

buffer G. Detection was done by incubation membranes with 1 ml

of CDP star substrate supplied with the kit for 3 min, 1020 rpm
at RT. Xray films (Kodak Scientific Imaging Film, Eastern Kodak
Company, Rochester, NY) were exposed to the membrane for
different time periods and films were developed using Kodak Image
developer. Xray images were scanned and converted to digital image
for further analysis.
Analysis. Image analysis was done using GEArray Expression
Analysis Suite (Superarray Bioscience, Frederic, MD). Gene densities
were expressed as average density (total density divided by number of
pixels). Background detection was done locally i.e., each expression
value was individually subtracted with value from the area outside
the capture grid but within the spot cell area. Data normalization was
done with non-modulating house keeping gene, Glyceraldehyde3
phosphate dehydrogenase (GAPDH). A gene was considered absent
where the average density of the spot was less than the mean value of
the local backgrounds of the lower 75th percentile of all non-bleeding
spots. All other spots were considered present.
Reverse Transcription (RT) and Polymerase Chain Reaction
(PCR). cDNA was synthesized using the Superscript first strand
synthesis system for RTPCR kit (Invitrogen Inc. Carlsbad, CA) as
per manufacturers instruction. The primers for AKT1 and cmyc
were previously described.13,14
Zymogram. Cells were seeded onto sixwell plates in the propogation media mentioned above. Cells were subsequently washed in
ultra culture Serum free media twice and the cells were then treated
with TPA (80 nM) and/or varying doses of EGCG and GTP for 24
in the serum free medium for an additional 24 hrs. The conditioned
media for different treatment were collected and standardized to cell
number and was concentrated five times, mixed with non reducing
sample buffer and were loaded on 10% Zymogram (Gelatin) gels
(invitrogen life technologies, Carlsbad, CA) and electrophoresis was
carried out in novex protein gel running tank (X Cell II, Novex
Experimental Technologies) at 110 volts for 3 h at 4C. Gels were
then taken out by breaking the cast, making sure that gels do not dry
1940

out, then washed with 2.5% triton X 100 for 30 min over shaker
(32 cycles/min) with one change of solution after 15 minutes
followed by the washing with double distilled water for 20 min over
shaker (32 cycles/min) with one change of water after 10 min. Gels
are then treated overnight with substrate buffer (Triscl: 50 mM:,
CaCl2 5 mM:, Tween 20: 0.02%) at 37C. followed by the staining
with 0.5% Comassie blue R250 solution (Comassie R250: 0.5%:,
Acetic Acid: 10%:, Methanol: 40%) for 1 h over shaker (32 cycles/
min). Subsequent destaining was done with destaining buffer (Acetic
Acid: Methanol: water:: 1:4:6) for 20 min with one change of buffer
after 10 min. Gels were than washed with double distilled water and
kept in distilled water overnight. The molecular weights (m.w.) of the
proteolytic bands were determined in relation to the reference marker
proteins, which were simultaneously loaded in the gel.
Cellular activation of signaling ELISA for AKT S473. Cellular
Activation of Signaling ELISA (CASE) Kit for AKT S473
(Superarray Bioscience, Frederic, MD) was used to analyse AKT
phosphorylation levels in Polyphenol treated cells according to
manufaturers protocol. This cellbased ELISA kit quantifies the
amount of activated (phosphorylated) AKT protein relative to total
AKT protein.
Nuclear extract. Nuclear extracts were prepared with a NEPER
Nuclear and Cytoplasmic Extraction Reagent (Pierce) according to
the manufacturers protocol. The levels of betacatenin in the cytoplasmic and nuclear extracts were assessed by immunoblotting with
anti bcatenin (Santa Cruz Biotechnology, Santa Cruz, CA).

Results
Effect of EGCG and GTP on DNA fragmentation. Typical
DNA ladder pattern of internucleosomal fragmentation is known as
a biological hallmark of apoptosis. To determine whether EGCG and
GTP induce apoptosis, DNA was isolated from treated and untreated
cells and was then fractionated by agarose gel electrophoresis. A
typical DNA ladder pattern was observed in cells treated with both
EGCG and GTP which was dose dependent, thus showing that both
EGCG and GTP treatment induce apoptosis in MDAMB231 cells
(Fig. 1).
Effect of EGCG and GTP on DNA proteins involved in apop
tosis. Western blot analysis showed that EGCG and GTP increased
bax and decreased bcl2 in a dose dependent manner (Fig. 2). After
treatment with EGCG and GTP for 24 h, the fulllength form of
the PARP protein (116 kDa), a substrate for caspase3, degraded into
the cleaved form (85 kDa) (Fig. 2). Thus we can say that EGCG and
GTP may induce apoptosis by altering the Bax/Bcl2 ratio favoring
apoptosis and upregulating proteases and inducing PARP cleavage.
EGCG and GTP treatment decreases MDAMB231 cell inva
sion through Matrigel in vitro. The effects of EGCG and GTP
on tumor cell invasion were investigated invitro by utilizing a
synthetic basement membrane system (a modified Boyden chamber).
Tumor cells were plated on a partition barrier, consisting of a
porous filter (8micron pores) which was coated with a reconstituted basement membrane matrix (Matrigel), and induced
to migrate across the barrier with medium enriched with 5%
FBS(chemoattractant). The cells that invaded matrigel cells were
fluorescence labeled with Calcein AM, which is a cellpermeant
dye that can be used to determine cell viability in most eukaryotic
cells. In live cells, the non-fluorescent calcein AM is converted to a

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Green Tea Induces Apoptosis and Inhibits Invasion

fluorescent calcein after acetoxymethyl ester hydrolysis by intra-

cellular esterases. Quantitative Fluroscence labeling resulted in
demonstrating a 2428% reduction in the invasion by EGCG and
1523% reduction by GTP in a dose dependent manner (Fig. 3).
Effect of EGCG and GTP on MMP9 expression. To compare
the differential gene regulation in polyphenol treated and untreated
cells, we did a focused microarray with the breast cancer biomarker
gene array panel. The analysis indicated more than fivefold down
regulation of MMP9 in both EGCG and GTP treated cells when
compared with the untreated controls (Fig. 3). To confirm the results
of the microarray experiment we did a reverse transcriptase PCR
reaction for the RNA isolated from cells treated and untreated with
the polyphenol. The results showed that MMP9 was downregulated
in the treated cells when compared with untreated thus confirming
the results of the microarray experiment (Fig. 3). Moreover the Figure 2. Effect of EGCG and GTP on proteins involved in apoptosis.
inhibition of MMP9 transcript was dose dependent (data not Representative picture of Western blot of cell cycle proteins of MDAMB231
shown). To compare MMP9 activities in cell supernatants from treated with 50 and 80 mg/ml of EGCG and 40 and 60 mg/ml GTP 24 hrs
from two independent experiments.
conditioned media from EGCG and GTP
treated and untreated cells were subjected
to gelatin zymography. The result as shown
in Figure 3 indicated that increasing doses
of EGCG and GTP caused gradual decrease
in MMP9 expression.
Effect of EGCG and GTP on AKT and
AKT phosphorylation. It has been shown
that constitutively active AKT protect cells
from apoptosis and also aids in invasion.
Western blot analysis using antiphosphospecificAkt antibody showed that both
EGCG and GTP suppressed AKT phosphorylation in a dose dependent manner
(Fig. 4). Cellular Activation of signaling
ELISA for AKT S473 also confirmed that
EGCG and GTP inhibited the AKT phosphorylation in a dose dependent manner
(Fig. 4). Furthermore EGCG and GTP
inhibited the AKT1 protein levels levels as
Figure 3. Effect of EGCG and GTP on invasion. (A) EGCG and GTP inhibits the invasive capacity of
revealed by Western blots (Fig. 5). Reverse MDAMB231 in vitro in a synthetic basement membrane system. Error bars indicate SEM (n = 4).
transcriptase PCR for AKT1 revealed a *Significantly different compared with controls, p < 0.05. (B) EGCG and GTP downregulates MMP9
dose dependent downregualtion of AKT1 transcript. (a) Microarray analysis, (b) Reverse Transcriptase Polymerase chain reaction. (C) MMP gelatigene in both EGCG and GTP treatments nolytic activity was performed with the conditioned media from MDAMB231cell culture. Data represent
(Fig. 5). AKT1 has been recently found to three individual experiments.
be a target gene for betacatenin. Cmyc is
a well known target gene of betacatenin induced transcription and
Discussion
was also found to follow the same pattern as AKT1 in EGCG and
SERM and Aromatase inhibitors are currently the popular treatGTP treated samples (Fig. 5).
Effect of EGCG and GTP on betacatenin. The activation of ment options for hormone dependent breast cancer. Resistance
Tcf regulated genes results from the accumulation of betacatenin eventually develops to all forms of treatment and hormone nonin the nucleus. We performed the western blot analysis for the responsive form of breast cancer is extremely difficult to manage as
cytoplasmic and nuclear fraction of cell lysates which was previ- it progresses to a metastatic stage disease. Hence it becomes pivotal
ously treated with EGCG and GTP for betacatenin. The amount to investigate other strategies to control tumor proliferation. In this
of betacatenin protein was reduced both in the cytoplasmic as well study, we investigated apoptotic and antiinvasive properties of GTP
as in the nuclear fraction in the treated group when compared to and EGCG in estrogen receptor negative MDAMB231. Our results
untreated control (Fig. 5). Therefore the data suggests that decreased suggest that EGCG induces apoptosis and reduces invasive propamount of betacatenin would have resulted in reduced formation erty of the aggressive MDAMB231. These data also suggest that
of betacateninTCF4 complex culminating in downmodulation of the beneficial properties of GTP and EGCG may be due to AKT
betacatenin/Tcf regulated genes like cmyc and AKT1.
inhibition.

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Green Tea Induces Apoptosis and Inhibits Invasion

Figure 5. Effect of EGCG and GTP on AKT. (A) Representative picture of

Western blot of AKT of MDAMB231 treated with 50 and 80 mg/ml of
EGCG and 40 and 60 mg/ml GTP for 24 hrs. (B) Western blot of cytoplasmic
and nuclear betacatenin of MDAMB231 treated with 50 and 80 mg/ml
of EGCG and 40 and 60 mg/ml GTP for 24 hrs. (C) Reverse Transcriptase
Polymerase chain reaction for cmyc and AKT1 of MDAMB231 treated with
50 and 80 mg/ml of EGCG and 40 and 60 mg/ml GTP for 24 hrs. GAPDH
is used as normalization control. Data is representative of two independent
observations.

Figure 4. Effect of EGCG and GTP on AKT Phosphorylation. (A) Representative

picture of Western blot of AKT and PhosphoAKT of MDAMB231 treated
with 50 and 80 mg/ml of EGCG and 40 and 60 mg/ml GTP for 24 hrs.
(B) Western blot of AKT and PhosphoAKT of MDAMB231 treated with 50
and 80 mg/ml of EGCG and 40 and 60 mg/ml GTP for 24 hrs. (C) Cellular
Activation of signaling ELISA for S473. Representative figure from two independent experiment.

Many of the molecular alterations that accompany carcinogenesis

lead to uncontrolled proliferation and the ability of transformed cells
to evade apoptosis. Both GTP and EGCG were pro apoptotic as
showed by intranucleosomal cleavage of DNA, which was visualized by a typical DNA ladder pattern. Induction of apoptosis was
primarily due to Bax/bcl2 ratio favouring apoptosis. PARP cleavage
was also observed in polyphenol treated groups. Metastasis plays
a major role in increasing the mortality of breast cancer patients
and phytoestrogens are found to reduce the metastatic potential of
breast cancer cells in vitro.15 Green tea polyphenol has recently been
showed to have antimetastatic effect on metastasisspecific mouse
mammary carcinoma 4T1 cells.16 We have shown the antiinvasive
property of EGCG and GTP in modified Boyden chamber in highly
invasive MDAMB231 breast cancer cell line. Invasion being a
prominent initial step in metastasis, antiinvasive capacity of these
1942

phytoestrogens might have implications in increasing the survival

time and decreasing morbidity of breast cancer patients.
For tumor angiogenesis as well as tumor invasion, degradation of
extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is
a critical process. Main function of MMPs is degradation of extracellular matrix contributing to cancer whereby tumor cells utilize the
matrix degrading capability of these enzymes to spread to distant
sites and additionally MMPs also are thought to promote growth
of these tumor cells once they have metastasized.17 Although many
MMPs have been identified, MMP2 (GelatinaseA) and MMP9
(GelatinaseB) are thought to be the key enzymes as they degrade
type IV collagen, main component of ECM.18,19 In our study
MMP9 was found to be downregulated at the transcriptional level as
revealed by focused microarray analysis, which was further confirmed
by reverse transcriptase polymerase chain reaction. Zymographs
of cell culture supernatants revealed that GTP and EGCG modulate gelatinolytic activities of MMP9 indicating that they may be
exerting their invasion inhibitory effect by inhibiting proteinases.
Thus we have shown that the polyphenols inhibit invasion of human
breast cancer cells which is consistent with recently published work20
and our work additionally delineate the molecular target as well.
PI3K/AKT signaling been found to be involved in survival and
proliferation of a variety of tumor cells and their hyperactivation
has resulted in altering the response of tumor cells to chemotherapy
and irradiation.21 Recent study shows AKT to be one of the major
pathway for MMP9 expression and revelead that isoginkgetin, a

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2007; Vol. 6 Issue 12

Green Tea Induces Apoptosis and Inhibits Invasion

biflavanoid for Dawn redwood to potently inhibit MMP9 expression

and tumor cell invasion through the inhibition of AKT pathway.22 In
our study, AKT was found to be inhibited both at the RNA and the
protein level by polyphenol treatment. Moreover, EGCG and GTP
decreased AKT phosphorylation as found out by western blotting.
Betacatenin signaling pathway contributes to transcriptional
regulation of AKT122 and hence AKT1 can be upregulated by a
dysregulated betacatenin signaling. Also betacatenin signaling is
activated in breast cancer and is upregulated by numerous pathways
in more than 50% of breast tumors.23 Thus, we investigated whether
betacatenin signaling is affected by EGCG and GTP. Betacatenin
protein level was decreased in cytoplasm and nucleus with polyphenol treatment. Cmyc, one of the prominent among betacatenin
regulated genes was also found to be downregulated by EGCG and
GTP treatment and followed same pattern as AKT1 suggesting
betacatenin can be involved in polyphenol mediated down regulation of AKT1.
These data provide a basic mechanism for chemotherapeutic
properties of EGCG and GTP in human breast cancer cells. Since
deregulation of AKT pathway has been implicated in cancer, investigation of molecular mechanisms that govern the expression of AKT
should provide insight into its biological functions and can be used
to design treatment strategies to target this pathway for prevention
and treatment of cancer. Future studies are planned to further dissect
the precise mechanism(s) of EGCG and GTP and to exploit its full
potential as breast cancer chemopreventive. Further invivo studies
are pivotal to confirm the translation of these invitro mechanisms
and are required to determine future therapeutic applications and
chemopreventive effects of EGCG and GTP against breast cancer.

16. Baliga MS, Meleth S, Katiyar SK. Growth inhibitory and antimetastatic effect of green tea
polyphenols on metastasisspecific mouse mammary carcinoma 4T1 cells in vitro and in
vivo systems. Clin Cancer Res 2005; 11:191827.
17. John A, Tuszynski G. The role of matrix metalloproteinases in tumor angiogenesis and
tumor metastasis. Pathol Oncol Res 2001; 7:1423.
18. Stamenkovic I. Extracellular matrix remodelling: The role of matrix metalloproteinases. J
Pathol 2003; 200:44864.
19. Hojilla CV, Mohammed FF, Khokha R. Matrix metalloproteinases and their tissue inhibitors direct cell fate during cancer development. Br J Cancer 2003; 89:181721.
20. Thyagarajan A, Zhu J, Sliva D. Combined effect of green tea and Ganoderma lucidum on
invasive behavior of breast cancer cells. Int J Oncol 2007; 30:9639.
21. Kuo PL, Hsu YL, Cho CY. Plumbagin induces G2M arrest and autophagy by inhibiting
the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol Cancer Ther
2006; 5:320921.
22. Dihlmann S, Kloor M, Fallsehr C, von Knebel Doeberitz M. Regulation of AKT1 expression by betacatenin/Tcf/Lef signaling in colorectal cancer cells. Carcinogenesis 2005;
26:150312.
23. Cowin P, Rowlands TM, Hatsell SJ. Cadherins and catenins in breast cancer. Curr Opin
Cell Biol 2005; 17:499508.

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